Polycythemia Vera: FURTHER IN VITRO STUDIES OF HEMATOPOIETIC REGULATION

Further in vitro studies of hematopoietic regulation were carried out in two patients with polycythemia vera who were also heterozygotes (Gd^B/Gd^A) for glucose-6-phosphate-dehydrogenase (G-6-PD). While only G-6-PD type A was detectable in circulating erythrocytes, granulocytes and platelets, cultures of peripheral blood and marrow from one patient revealed a substantial number of G-6-PD type B erythroid burst-forming units (BFU-E) and granulocyte/macrophage colony-forming units. Detailed analysis demonstrated: (a) where detectable, normal BFU-E and granulocyte/macrophage colony-forming units were found with similar frequencies; (b) the same frequencies for normal progenitors characterized cultures of peripheral blood and marrow; (c) inhibition of normal erythroid differentiation between BFU-E and the more mature erythroid colony-forming unit; (d) a decline in the prevalence of normal colony-forming units with time, suggesting that disease progression is associated with further suppression of normal hematopoiesis by products of the abnormal clone.     

Feline glucose-6-phosphate dehydrogenase cellular mosaicism. Application to the study of retrovirus-induced pure red cell aplasia.

Neoplasms result from the uncontrolled proliferation of abnormal or transformed cells. The early stages of this process are difficult to study because of the lack of sensitive and specific markers of clonal evolution in an experimental system. We have developed a cat model using cellular mosaicism for glucose-6-phosphate dehydrogenase (G-6-PD). Our findings confirm that the structural locus for feline G-6-PD is on the X-chromosome and demonstrate that it is randomly inactivated in somatic cells. Heterozygous cats have balanced ratios of G-6-PD enzyme types in peripheral blood cells and hematopoietic progenitors that remain stable over time. In our initial studies, we used the model to analyze the events surrounding marrow failure experimentally induced by selected strains of feline leukemia virus (FeLV). Two G-6-PD heterozygous cats, one F1 male hybrid and one domestic cat were infected with FeLV (C or KT) and developed pure red cell aplasia (PRCA). Colonies arising from the more mature erythroid colony-forming cell were not detected in marrow culture of anemic animals although erythroid bursts persisted, suggesting that the differentiation of early erythroid progenitors (BFU-E) was inhibited in vivo. The ratio of G-6-PD types in hematopoietic progenitors and peripheral blood cells from the heterozygous cats did not change when the animals developed PRCA. Thus, the anemia did not result from the clonal expansion of a transformed myeloid stem cell. With this experimental approach, one may prospectively assess clonal evolution and cellular interactions in other FeLV-induced diseases.     

Platelet glucose-6-phosphatase activity in patients with von Gierke's disease and their parents

The present report describes the results of an assay of platelet glucose-6-phosphatase (G-6-Pase) in patients with von Gierke's disease and their parents. The G-6-Pase activity of the patients was found to be about 10–25% of that of controls, while that of the parents showed an intermediate value between that of the patients and that of the controls, which led to the conclusion that the parents were the heterozygotes of the disease. These results suggest a possible method for the diagnosis of G-6-Pase deficiency as well as for detection of the heterozygotes of the disease by a platelet G-6-Pase assay without making a G-6-Pase assay of liver biopsy materials.As for the separation of platelets, a differential centrifugation was undertaken using Na₂ -EDTA as an anticoagulant. In the G-6-Pase assay, universally ¹⁴C-labelled glucose 6-phosphate ([U-¹⁴C]-G-6-P) was used as a substrate and the enzyme activity was measured by the amount of glucose liberated by the platelet suspension after the reaction. The effect of non-specific phosphatase was estimated by using universally ¹⁴C-labelled glucose 1-phosphate ([U-¹⁴C] -G -6-P) as substrate.In the diagnosis of von Gierke's disease, as well as the detection of the heterozygotes of the disease by the G-6-Pase assay using platelets as material, the effect of non-specific phosphatase was considered to be negligible.     

Glucose-6-phosphate dehydrogenase activity in term and near-term, male African American neonates

BACKGROUND: We determined values for glucose-6-phosphate dehydrogenase (G-6-PD) activity in African American neonates. METHODS: G-6-PD activity was measured on umbilical cord blood from term and near-term healthy, male neonates. Neonates were stratified according to the number of neonates for each numerical unit of G-6-PD activity. Corrected end tidal carbon monoxide (ETCOc), a non-invasive index of hemolysis, was performed on each neonate. At least one predischarge transcutaneous bilirubin determination was performed. RESULTS: Five hundred neonates were studied. Two subpopulations were apparent, with no overlap between the subgroups. Mean value for the 64 (12.8%) infants with the lower values (G-6-PD deficient) was 2.7+/-1.1 U/g Hb, range 0.4-6.6 U/g Hb, while that for the 436 neonates with the higher values (G-6-PD normal) was 21.8+/-2.9 U/g Hb, range 14.5-33.8 U/g Hb. No significant differences in activity were noted between those neonates <37 weeks gestational age and those >37 weeks. Enzyme activity in the lower range in both groups was not related to the development of hyperbilirubinemia. G-6-PD enzyme activity did not correlate with ETCOc values either for the entire cohort or for the individual subsets. CONCLUSIONS: G-6-PD-deficient neonates formed a separate subgroup from those with normal enzyme activity. The data supplied should facilitate interpretation of G-6-PD test results.     

Evidence for an effect on erythrophilic IgG-globulin coat by the haemolytic process

Erythrophilic IgG globulin coat (IgG E-C) is a fraction separated from serum IgG-globulin by cellulose phosphate column chromatography. It is easily eluted from the red blood cells and may be quantitated by a method described in this paper. This method was applied to 20 completely healthy newborns and to 81 newborns with severe jaundice of various aetiology (i.e. ABO isoimmunisation, haemolysis severe; ABO isoimmunisation, haemolysis mild; G-6-PD deficiency, haemolysis severe; G-6-PD deficiency, haemolysis mild; ABO incompatibility; Rh isoimmunisation and unknown aetiology). It was also applied to 19 children with favism and 10 age matched symptomless children with G-6-PD deficiency. IgG E-C mean values were higher in newborns with severe jaundice due to ABO isoimmunisation or G-6-PD deficiency as compared to those with jaundice of other aetiology or the healthy controls. The difference was even more pronounced in cases with severe haemolysis (0.01 > p > 0.001). They were also higher in children with G-6-PD deficiency only during the crisis of favism (0.01 > p > 0.001). It is concluded that IgG E-C increases during acute haemolysis.     

Evaluation of glucose-6-phosphate dehydrogenase activity in two different ethnic groups using a kit employing the haemoglobin normalization procedure

AIM: Correct evaluation of Glucose-6-Phosphate Dehydrogenase (G-6-PD) activity of two ethnic groups using a fully quantitative kit with a simultaneous Hemoglobin Normalization (Hb Normalization) procedure. DESIGN AND METHODS: Two groups of mothers and their healthy full term newborns of Greek (n = 1.166) and Albanian (n = 818) origin were tested for their G-6-PD activity employing a direct normalization protocol. RESULTS: Greek mothers and newborns showed a higher prevalence for G-6-PD deficiency as compared to those of Albanian origin. Males of G-6-PD deficient mothers confirmed the efficacy of the method. CONCLUSION: A fully quantitative G-6-PD kit employing Hb Normalization is essential for the correct classification of G-6-PD activity, both in male and female subjects.     

Intravascular hemolysis in aluminium phosphide poisoning.

Intravascular hemolysis is most often secondary to exposure to a variety of drugs or infections, and usually occurs in patients who are deficient in glucose-6-phosphate dehydrogenase (G-6-PD) enzyme. Aluminium phosphide, a fumigant widely used in India, has been reported to produce intravascular hemolysis in only one patient who also had concomitant G-6-PD deficiency. This report describes the occurrence of intravascular hemolysis with aluminium phosphide poisoning in a patient with normal G-6-PD levels. This is of significance as jaundice in patients with this poisoning is often attributed to hepatic damage alone.     

Microheterogeneity of glucose-6-phosphate dehydrogenase phenotypes

Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme of the pentose phosphate pathway and has been extensively investigated. Several variants of G-6-PD were encountered in different regions of Saudi Arabia during an extensive screening programme. A modified rapid isoelectric focussing procedure using LKB Ampholine PAG plates (pH 5.5-8.5) was used to separate the isoenzymes of G-6-PD. G-6-PD-B+ (the normal enzyme) was separated into five major and three minor fractions. The pattern obtained for G-6-PD-A+, G-6-PD-A-, G-6-PD-Mediterranean and G-6-PD-Mediterranean-like showed that the variants differ significantly in their isoenzyme pattern. In this paper it is shown that isoelectric focussing is a sensitive, powerful and reproducible technique to study microheterogeneity of red cell enzymes.     

Characteristics and significance of the reverse glucose-6-phosphate dehydrogenase reaction

Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme of the hexose monophosphate pathway, and this important reaction is often considered to be irreversible. However, its apparent irreversibility is caused by the rapid removal of the immediate product, 6-phosphoglucono-delta-lactone. We have now investigated the reverse G-6-PD reaction, namely, the oxidation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) by 6-phosphoglucono-delta-lactone to form glucose-6-phosphate and nicotinamide-adenine dinucleotide phosphate (NADP). The substrate of the reaction, 6-phosphoglucono-delta-lactone, was rapidly generated from glucose-6-phosphate and NADP. The lactone was stabilized by addition of perchloric acid. A substrate analogue, 6-phosphoglucono-gamma-lactone, was synthesized by dehydrating 6-phosphogluconic acid. At pH 2.3 the t 1/2 of the delta-lactone was 2.4 hours; that of the gamma-lactone was 57 hours. The following kinetic parameters were established: Km delta-lactone 1027 +/- 183 mumol/L; Km gamma-lactone 266 +/- 71 mumol/L; Km NADPH less than 10 mumol/L; ratio of the Vmax G-6-PD forward/reverse reaction 2.0. Glucose-6-phosphate was found to be a competitive inhibitor with both lactones in the reverse G-6-PD reaction. Genetic mutants of humans in which the Km of G-6-PD for glucose-6-phosphate was diminished also had a diminished Km for 6-phosphoglucono-delta-lactone. Thus, it appears that the same active site on the enzyme binds glucose-6-phosphate in the forward reaction and 6-phosphogluconolactone in the reverse reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic diversity of the “Mediterranean” glucose-6-phosphate dehydrogenase deficiency phenotype

Genetic diversity of the "Mediterranean" phenotype of G-6-PD (glucose-6-phosphate dehydrogenase) deficiency was revealed when detailed studies were performed on blood specimens from 79 Greek males with G-6-PD levels 0-10% of normal. Four different mutants were found to be responsible for the severely deficient phenotypes: two mutants. G-6-PD U-M (Union-Markham) and G-6-PD Orchomenos, were distinguishable by electrophoresis, while the other two. G-6-PD Athens-like and G-6-PD Mediterranean, were distinguishable on the basis of their kinetic characteristics. Of the kinetic tests applied, the most useful for differentiating the variants were those measuring utilization rates of the analogue substrates deamino-NADP, 2-deoxyglucose-6-phosphate, and galactose-6-phosphate. Among unrelated males with severe G-6-PD deficiency, the relative frequencies of the four variants were: G-6-PD U-M. 5%; G-6-PD Orchomenos, 7%; G-6-PD Athens-like, 16%; G-6-PD Mediterranean, 72%. Genetic, biochemical, and clinical implications of the findings are discussed.     

Polycythemia vera. The in vitro response of normal and abnormal stem cell lines to erythropoietin.

Bone marrow cells from two glucose-6-phosphate dehydrogenase (G-6-PD) heterozygotes with polycythemia vera were cultured to determine whether progenitors which wre not of the polycythemia vera clone were present, and, if present, which cell lines contributed to the increase in erythroid colonies observed in response to added erythropoietin (ESF). To accomplish this, the G-6-PD isoenzyme activity of individual erythroid colonies was determined. All of the erythroid colonies analyzed in cultures without added ESF, contained the G-6-PD isoenzyme type characteristic of the abnormal clone. With higher ESF concentrations in the culture, however, there was an increase in the colonies that were not of the polycythemia vera clone. Analysis of the ratio of the various types of colonies indicated that normal and polycythemia vera cells are capable of responding to ESF in vitro. In selected patients, this technique permits analysis of the ratios of normal to abnormal cells during the course of the disease, in response to therapy and during late complications, such as myelofibrosis or leukemic transformation.     

Detection of glucose-6-phosphate dehydrogenase deficiency in erythrocytes: a spectrophotometric assay and a fluorescent spot test compared with a cytochemical method

The results of a quantitative spectrophotometric enzyme assay, a fluorescent spot test and a cytochemical assay for glucose-6-phosphate dehydrogenase deficiency were compared systematically. The high sensitivity of the spectrophotometric assay and the fluorescent spot test in the detection of severely deficient individuals was confirmed. For the detection of heterozygote females, however both tests were unreliable; the sensitivities of the fluorescent spot test and the spectrophotometric assay being 32% and 11% respectively. Specificities for both tests were high (99%). Introduction of the ratio of glucose-6-phosphate dehydrogenase and pyruvate kinase (G-6-PD/PK ratio) activities increased the sensitivity of the spectrophotometric assay to nearly 100%. It is concluded that the fluorescent spot test should be used for the diagnosis of G-6-PD deficiency in developing countries; whereas if spectrophotometric enzyme assays are available, the G-6-PD/PK ratio should always be performed. In cases where the ratio is less than 0.70, cytochemical analysis is indicated.     

葡萄糖-6-磷酸脱氢酶活性浓度检测法的初步研究

目的建立一种简单、快速、可自动检测葡萄糖-6-磷酸脱氢酶（G-6-PD）在血红蛋白（Hb）中活性的方法。方法用自配的检测试剂，在全自动生化分析仪上检测抗凝血标本的G-6-PD活性，同时检测Hb浓度，并计算出G-6-PD在Hb中的活性浓度（U／gHb），对同一标本用简易快速高铁血红蛋白还原试验、G-6-P／6-GP比值法进行对比试验。结果G-6-PD活性浓度的参考范围为≥5．0U／gHb，G-6-PD活性浓度检测法与G-6-P／6-GP比值法及简易快速高铁血红蛋白还原试验检测法的检测结果具有高度一致性。G-6-PD活性浓度检测法可用不洗涤红细胞，其检测试剂及检测方法具有良好的重复性。制备溶血液时吸样量从7～13μl的G-6-PD活性浓度检测结果差异无统计学意义（P〉0．05）。直接用酸性枸橼酸右旋糖（ACD）抗凝全血比用压积红细胞检测G-6-PD活性浓度的结果高。结论G-6-PD活性浓度检测法是一种简单、快速、重复性好、操作简便的检测方法。     

Acetylsalicylic acid-induced hemolysis and its mechanism

Acetylsalicylic acid (ASA) is known to cause severe hemolytic anemia in some glucose-6-phosphate-dehydrogenase-deficient (G-6-PD-deficient) individuals. To study its mechanism, erythrocytes from an ASA-sensitive patient were transfused into a normal compatible recipient. The administration of 2,5-dihydroxybenzoic (gentisic) acid, a known ASA metabolite with redox properties, to the recipient resulted in a marked decrease in the survival of the patient's erythrocytes. Similar studies with red cells from individuals with A- and Mediterranean variants of G-6-PD revealed no alteration in the erythrocytes' survival. Further studies disclosed that both salicylate and gentisate competitively inhibited the G-6-PD from the ASA-sensitive patient resulting in a marked change in the K(m) for NADP. These drugs also inhibited the A- and Mediterranean variants of G-6-PD. The magnitude of inhibition, however, was comparatively small and not different from that observed with a normal enzyme.The above studies suggested that enzyme inhibition by salicylate and gentisate may play an important role in ASA-induced hemolysis. Such an inhibition would further curtail NADPH regeneration, rendering the cells more vulnerable to oxidants. In this connection, gentisate seems to play a major role in ASA-induced hemolysis for it is both a G-6-PD inhibitor and an "oxidant."     

Polycythemia vera. Increased expression of normal committed granulocytic stem cells in vitro after exposure of marrow to tritiated thymidine.

In previous studies of two patients with polycythemia vera (PV) and heterozygous at the X-linked locus for glucose-6-phosphate dehydrogenase (G-6-PD), only type A isoenzyme was found in non-lymphoid hematopoietic cells. However, some granulocytic and erythrocytic colonies grown in vitro had type B G-6-PD and therefore arose from presumably normal progenitors. In this study we exposed marrow cells from these same two patients to high-specific activity tritiated thymidine (3HTdR) before culture to kill cells actively synthesizing DNA. Individual granulocytic colonies were plucked and tested for G-6-PD after 14 d of culture. The frequency of type B colonies rose after exposure to 3HTdR from 8/101 to 11/36 in patient 1 and from 0/32 to 6/31 in patient 2 (P less than 0.003). No increase in the frequency of normal erythroid bursts after 3HTdR exposure was seen, implying that in PV, early granulopoiesis, and erythropoiesis are regulated differently. The results demonstrated that only type A granulocytic colonies, arising from the abnormal clone, were removed by the 3HTdR. In addition, for patient 2, statistical analysis indicated there was an absolute increase in normal granulocytic colonies detected in culture. Thus, PV clonal colony-forming units in culture (CFU-C) cycle more rapidly than do normal CFU-C and may suppress proliferation of normal CFU-C in vitro.     

Pancytopenia as a clonal disorder of a multipotent hematopoietic stem cell

Hematopoiesis was investigated in a 14-yr-old girl who had a 2-yr history of stable asymptomatic pancytopenia and who was also heterozygous at the structural locus for glucose-6-phosphate dehydrogenase (G-6-PD). There was no morphologic or cytogenetic evidence for preleukemia and no suggestion of Fanconi anemia. In the skin and sheep erythrocytes-rosetted T lymphocytes, the ratio of G-6-PD A/B activities was 1:1. However, only type B activity was found in peripheral blood erythrocytes, granulocytes, and platelets. Most erythroid bursts and all granulocyte/macrophage colonies formed in methylcellulose culture were derived from the abnormal clone. These findings demonstrate that (a) some cases of pancytopenia are stem cell diseases that apparently develop clonally; (b) circulating differentiated cells originate from this clone; (c) despite a hypoproliferative anemia, the in vivo expression of presumably normal (nonclonal) progenitors is suppressed. In this patient, the relationship between clonal dominance and possible malignancy may be assessed prospectively.     

新生儿葡萄糖-6-磷酸脱氢酶缺乏症的筛查与分析

目的通过对新生儿葡萄糖-6-磷酸脱氢酶（glucose-6-phosphate dehydrogenase,G-6-PD）缺乏症的筛查及临床分析,了解桂平市新生儿G-6-PD缺乏情况及G-6-PD缺乏幼儿与高胆红素血症的相关性。方法对2010年7月至2011年7月在该院出生的3 402例新生儿进行G-6-PD测定,统计分析G-6-PD缺乏情况,并跟踪回访新生儿至出生后第10天,如果出现黄疸应立即采血测定胆红素,对新生儿高胆红素血症的发生率进行比较分析。结果新生儿G-6-PD缺乏症发病率为10.35%（352/3 402）,男婴G-6-PD缺乏率为14.28%（264/1 849）;女婴G-6-PD缺乏率为5.66%（88/1 553）。G-6-PD缺乏幼儿高胆红素血症的发生率为25.3%,明显高于G-6-PD正常新生儿的2.6%（P〈0.01）。结论广西桂平市是G-6-PD缺乏症的高发区,对G-6-PD缺乏症的筛查非常有意义,能有效提示医生谨慎用药和指导患儿避免接触诱因,预防溶血的发生,提高该地区人群的健康素质。     

MSCT容积重建评价盆腔起源不明肿瘤的供血动脉

目的 采用多排螺旋CT血管造影术(MSCTA)容积重建(VR)技术评价盆腔起源不明肿瘤的供血动脉、判断肿瘤的来源、性质.方法 对37例盆腔起源不明肿瘤患者行MSCTA,采用血管生成和图像融合法重建腹部血管,通过观察肿瘤供血动脉的来源、数量、形态、起止、走行、分布、起源,初步判断肿瘤的良恶性及肿瘤是否侵犯周围脏器,并与手术结果进行对照分析.结果 MSCTA显示盆腔起源不明肿瘤的供血动脉63支,其中卵巢动脉15支(13例,均为卵巢肿瘤),子宫动脉35支(3例子宫肿瘤、15例卵巢肿瘤),肠系膜下动脉6支(2例结肠癌、3例肠道间质瘤),骶正中动脉2支(2例神经源性肿瘤),髂总动脉无名分支2支(1例淋巴瘤,1例肿瘤来源不明),腹主动脉其他分支3支,定位诊断准确率为91.89% (34/37),术前MSCTA诊断盆腔良、恶性肿瘤与手术结果符合率为89.19%(33/37).双侧病变5例,其中3例为原发,2例为继发,MSCTA诊断准确率为100%.结论 MSCTA VR技术可显示盆腔肿瘤的供血动脉,对盆腔起源不明的肿瘤定位、定性诊断有较高价值.     

Glucose-6-Phosphate Dehydrogenase (G-6-PD) Hamburg, a New Variant with Chronic Nonspherocytic Haemolytic Anaemia

Abstract. A new variant of G-6-PD with chronic nonspherocytic haemolytic anaemia and very low activity, named G-6-PD Hamburg, was partially purified and biochemically characterized. It was found to have very high lability, an unusually high Km for G-6-P (2000 μM), increased utilization rates for 2-desoxy G-6-P (133%) and galactose-6-phosphate (87%) and an abnormal pH-activity curve. The electrophoretic mobility seemed to be normal. The leukocytes also revealed diminished G-6-PD activity. No impairment of bactericidal activity of neutrophilic granulocytes, as shown by a normal nitroblue tetrazolium reduction, could be demonstrated.     

A modification of the serum glucose-6-phosphatase assay

The difficulties in estimating G-6-Pase activity (distinct from non-specific acid and alkaline phosphatases) in serum are discussed. Experiments with pregnant human serum and with rat liver microsomes suggest that G-6-Pase activity can be estimated specifically in serum buffered to pH 6.5, provided 1.4 × 10⁻³ BeSO₄ is added as an inhibitor of alkaline phosphatase. A second portion of the same serum, preincubated at pH 5.0 to destroy G-6-Pase activity, is used to estimate acid phosphatase activity. Subtraction of this from the first value results in an estimate of true G-6-Pase activity.